The long-term objective of this project is to understand the relation between DNA replication and chromatin assembly. This grant proposes to examine the biochemical mechanism of DNA replication-coupled chromatin assembly as well as to investigate the biological function of chromatin assembly factors in Drosophila melanogaster. This proposal seeks continued support of ongoing studies of Drosophila chromatin assembly factor-1 (dCAF-1) and a newly-discovered activity, replication-coupling assembly factor (RCAF). Both dCAF-1 and RCAF are specifically required for the assembly of newly-replicated DNA (relative to unreplicated DNA) into chromatin in an in vitro SV40 DNA replication assay. It is possible that RCAF is a key molecule that 'tags' newly-replicated DNA for chromatin assembly as well as for other important functions of DNA during progression through S phase. The Specific Aims are as follows. 1. Cloning and analysis of Drosophila chromatin assembly factor 1 (dCAF- 1). The cDNAs encoding three of the four polypeptides that comprise dCAF-1 have been isolated. The objective of this aim is to clone the remaining subunit of dCAF-1 and then to proceed onto the characterization of the biochemical properties and biological functions of dCAF-1. 2. Purification and cloning of Drosophila replication-coupling assembly factor (RCAF). The objectives of these experiments are the purification of RCAF, isolation of the cDNA(s) encoding RCAF, and the synthesis and purification of recombinant RCAF. 3. Characterization of RCAF in vitro and in vivo. The objective of these studies is to analyze the biochemical mechanism by which RCAF participates in DNA replication-coupled chromatin assembly as well as the biological function of RCAF in vivo in Drosophila. These experiments would contribute to our understanding of the formation of chromatin as well as the role of chromatin in processes such as transcription, replication, repair, and recombination. Thus, these studies should be directly applicable to the analysis of human disease, including many forms of cancer, that involve defects in these important chromatin- utilizing processes.